Prostanoids, including prostaglandin (PG), prostacyclin and thromboxane (TX), are important mediators of central and peripheral physiological effects. Prostaglandin D2 (PGD2) is formed in a variety of tissues including brain, spleen, lung, bone marrow, stomach, skin and also in mast cells (Lewis et al., 1982). PGD2 has been implicated in many physiological events both in the central nervous system and in the peripheral tissues. In the central nervous system, PGD2 has been shown to affect the induction of sleep, body temperature, olfactory function, hormone release and nociception. Peripherally, PGD2 is the major cyclooxygenase product of arachidonic acid produced from mast cells following immunological challenge. Local allergen challenge in patients with allergic rhinitis, bronchial asthma, allergic conjunctivitis and atopic dermatitis has been shown to result in rapid elevation of PGD2 levels in nasal and bronchial lavage fluids, tears and skin chamber fluids. Activated mast cells, a major source of PGD2, are one of the key players in driving the allergic response in conditions such as asthma, allergic rhinitis, allergic conjunctivitis, allergic dermatitis and other diseases (Brightling et al., 2003). Likewise, PGD2 has many inflammatory actions, such as increasing vascular permeability in the conjunctiva and skin, increasing nasal airway resistance, airway narrowing and eosinophil infiltration into the conjunctiva and trachea. Therefore, PGD2 is considered one of the key players in driving inflammatory reactions.
Early efforts have focused on identifying distinct receptors for the five naturally occurring bioactive prostanoids, PGD2, PGE2, PGF2α, PGI2 and TXA2, resulting in the classification of five basic types of prostanoid receptors: DP, EP, FP, prostacyclin (IP) and thomboxane (TP) receptors, respectively (Coleman et al., 1994). Many of the actions of prostaglandin D2 are mediated through its action on the D-type prostaglandin receptor (DP), a G protein-coupled receptor. While originally thought that each prostanoid acted preferentially on individual receptors, researchers studying prostanoid biology have begun to appreciate the promiscuity of these ligands to interact with members of the different receptor families. Thus, it is becoming ever more clear that to understand prostanoid signaling one must elucidate the biological consequence of prostanoid receptor activation.
The DP receptor is of particular interest because it is found in both central and peripheral cells suggesting its involvement in mediating varied biological pathways and, consequently, its potential therapeutic importance in many disease states. DP receptors have been identified in the brain and PGD2 has effects on sleep induction, body temperature, olfactory function, and hormone release (Negishi, et al., 1993; Wright et al., 1999 and references within). DP receptors have also been localized to discrete and distinct cell populations of the spinal cord. This observation may explain the discordant effects of hyperalgesia and allodynia (discomfort from innocuous tactile stimuli) induced by PGD2. DP receptors are also present in the gastrointestinal tract and have been implicated in the contractile response of the GI tract (Wright et al., 1999; Ito et al., 1989). Additionally, DP receptor ligands have been shown to induce mucous secretion and cell proliferation of intestinal cells. Glycogenesis in the liver may also be regulated by DP receptors (Ito et al., 1989). DP receptors are found in the eye and agonists reduce intraoccular pressure suggesting a role in glaucoma. Platelets contain the DP receptor and PGD2 has been shown to ingibit platelet aggregation supportion a role for the DP receptor in modulating blood disorders such as thrombosis (Armstrong, 1996). Thus, the varied expression of the DP receptor in different organs and tissues suggests the DP receptor may be an attractive target for different therapeutic areas.
Of particular interest, the DP receptor has been implicated in various inflammatory disorders including but not limited to asthma, allergic rhinitis, airway hyperactivity, allergic dermatitis, allergic conjunctivitis and chronic obstructive pulmonary disease. This is supported by the observation that PGD2 is the major prostanoid released by immunochallenged mast cells (Roberts, et al., 1980). In asthma, the respiratory epithelium has long been recognized as a key source of inflammatory cytokines and chemokines that drive the progression of the disease (Holgate et al., 2000). In an experimental murine model of asthma, the DP receptor is dramatically upregulated on airway epithelium on antigen challenge (Matsuoka et al., 2000). Conversely, in knockout mice lacking the DP receptor, there is a marked reduction in airway hyperreactivity and chronic inflammation (Matsuoka et al., 2000); two of the cardinal features of human asthma. Similarly, DP receptor antagonists have been shown to reduce airway inflammation in a guinea pig experimental asthma model (Arimura et al., 2001). The DP receptor is also thought to be involved in human allergic rhinitis, a frequent allergic disease that is characterized by the symptoms of sneezing, itching, rhinorea and nasal congestion. Local administration of PGD2 to the nose causes a dose dependent increase in nasal congestion (Doyle et al. 1990). DP antagonists have been shown to be effective at alleviating the symptoms of allergic rhinitis in multiple species, and more specifically, have been shown to inhibit the antigen-induced nasal congestion, the most manifest symptom of allergic rhinitis. DP antagonists are also effective in experimental models of allergic conjunctivitis and allergic dermatitis (Arimura et al., 2001). Thus, DP antagonists could therefore be useful in the treatment of a variety of PGD2-mediated disorders including, but not limited to, bronchial asthma, Chronic obstructive pulmonary disease (COPD), allergic rhinitis, allergic dermatitis, allergic conjunctivitis, systemic mastocytosis and ischemic repurfusion injury.
Thus far, the DP receptor has been cloned from human (Boie et al., 1995), rat (Wright et al., 1999) and mouse (Hirata et al., 1994). These DP receptors share 73–90% homology at the amino acid level between human, mouse and rat and, in each case, activation of the recombinant receptors leads to accumulation of intracellular cAMP. It is generally observed between G protein coupled receptors that compounds often show varying potencies from one orthologue receptor to another.
Disclosed here, for the first time, is a DP receptor from the guinea pig. The present invention provides several advantages over that which is currently known in the art. Species differences between mouse, rat, human and guinea pig can now be more fully determined and characterized. The low expression levels of the DP receptor in native tissues makes it difficult to assess a compound's activity as a modulator, effector, agonist or antagonist of the receptor. The present invention now provides the opportunity to examine the receptor in an isolated and purified condition providing the ability to test compounds and then to bridge in vitro studies to the same species in vivo. Because of its larger size, the guinea pig is a preferred animal model to smaller rodents, for instance, providing more surface area with regard to dermatology and gastrointestinal studies. More importantly, the guinea pig is the most usable small animal model for some allergy models such as nasal congestion and is more responsive to airway hyperactivity manipulations. Although the guinea pig represents an ideal preclinical model for the evaluation of DP receptor modulators in multiple disease models, as outlined above, the cloning of the guinea DP receptor has not previously been reported and hence it is difficult to predict the affinity of a compound against this orthologue.